tory system and do not produce antibodies, SAR shares several common characteristics with the innate immune system of animals.
A substantial body of evidence indicates that salicylic acid (SA) is a critical signaling molecule in the pathway(s) leading to local and systemic disease resistance, as well as PR expression (10, 11). In addition, recent studies have demonstrated that ethylene and jasmonic acid (JA) mediate the activation of various defense responses and resistance to certain pathogens (12, 13). The relationship between the SA, ethylene, and JA signaling pathways is not well defined. SA has been shown to work synergistically with ethylene or the JA derivative methyl jasmonate to activate PR expression in tobacco and Arabidopsis (14, 15); however, other defense responses appear to be regulated by ethylene-and/or JA-dependent pathways that are independent of SA (16, 17).
Another signaling molecule that has been implicated in the activation of plant defenses is nitric oxide (NO). This compound has previously been shown to serve as a key redox-active signal for the activation of various mammalian defense responses, including the inflammatory and innate immune responses (18, 19). In contrast to the extensive studies during the past decade or more on NO's role in animal defense, only recently has NO's involvement in the plant defense response to pathogens been addressed (20). To date, NO's participation has been documented in at least three different plant-pathogen systems (21, 22). Current evidence suggests that there are many parallels between NO action in plants and animals.
In this paper, we will review some of our findings concerning NO-and SA-mediated signaling in plants. Particular emphasis will be placed on the results generated over the past few years. In addition, new evidence implicating NO and its second messenger cyclic ADP ribose (cADPR) in tobacco PR-1 gene activation is presented.
SA-Interacting Proteins in Tobacco. To clarify the mechanism(s) through which SA activates plant defense responses, we have sought to identify the effector(s) with which SA interacts. The first protein shown to reversibly bind SA was a catalase from tobacco, originally termed SABP, for SA binding protein (23). Catalases convert H2O2 to H2O and O2; this activity was inhibited by SA both in vitro and in vivo. Catalase activity was also inhibited by the synthetic SA functional analogs 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH), as well as by various biologically active SA analogs, which are capable of inducing PR expression and enhancing disease resistance (23, 24 and 25). By contrast, biologically inactive analogs of SA and INA failed to inhibit catalase activity (23, 24). Interestingly, H2O2 and various prooxidants induced PR-1 expression in tobacco whereas several antioxidants suppressed the SA-, INA-, or BTH-mediated activation of this gene (23, 25). Thus, SA was proposed to activate PR expression by increasing the levels of H2O2 and other ROS, which could then serve as second messengers in the defense signaling pathway.
Analyses of ascorbate peroxidase (26), the other major H2O2-degrading enzyme in plant cells, provided additional support for this hypothesis. SA, INA, and BTH were all found to inhibit ascorbate peroxidase activity whereas inactive analogs of SA did not (25, 26). However, different studies from both our lab (27, 28) and others (29, 30 and 31) have suggested that H2O2 functions upstream rather than, or in addition to, downstream of SA in the defense signaling pathway. Thus, the role played by SA-mediated catalase inhibition and elevated ROS levels in the induction of defense responses is currently unclear.
Another mechanism through which SA-mediated inhibition of catalase and ascorbate peroxidase might activate defenses is via the generation of SA free radicals. SA has been shown to inhibit catalase by serving as a one-electron donating substrate (32). In this process, SA is converted into a free radical, which could then initiate lipid peroxidation. SA and its biologically active analogs have been shown to induce lipid peroxidation in tobacco suspension cells (33). Moreover, exogenously applied lipid peroxides (the products of lipid peroxidation) induce PR expression in suspension cells.
In addition to catalase, a second SA-binding protein (SABP2) has been identified. This soluble, low molecular weight protein exhibits an affinity for SA (Kd = 90 nM) that is approximately 150 times higher than that of catalase (34). Strikingly, SABP2's affinity for BTH is 15-fold higher than its affinity for SA; this is consistent with BTH's greater efficacy at inducing PR expression and SAR.
Mitogen-Activated Protein (MAP) Kinases and the Activation of Defense Responses in Tobacco. Protein phosphorylation and/or dephosphorylation are known to play important roles in many signal transduction pathways. Based on studies using protein kinase and phosphatase inhibitors, at least two phosphoproteins were implicated in the SA signaling pathway of tobacco (35). One of these proteins appears to work upstream of SA whereas the other works downstream. A 48-kDa kinase that is rapidly and transiently activated by SA treatment was subsequently identified in tobacco suspension cells (36). Sequence analysis of a cDNA clone encoding this SA-induced protein kinase (SIPK) revealed that it is a member of the MAP kinase family. In addition to SA, SIPK was shown to be activated at the enzyme level by various pathogen-associated stimuli, including two elicitins and a cell wall-derived (CWD) carbohydrate elicitor from Phytophthora spp. (37), and bacterial harpin (ref.38; Table 1). SIPK also was activated in a gene-for-gene specific manner by the Avr9 peptide from Cladosporium fulvum in transgenic tobacco expressing the cognate resistance gene Cf-9 (39) and in tobacco mosaic virus (TMV)-infected Xanthi nc carrying the cognate resistance gene N (40). Additionally, SIPK was activated to high levels by wounding (41). Thus, this kinase appears to be involved in multiple signaling pathways.
The discovery that SIPK encodes a 48-kDa kinase that is strongly activated by wounding raised questions as to the identity of the wounding-induced protein kinase (WIPK) gene product. Transcripts of WIPK were shown to accumulate after wounding stress (42). This observation led to the hypothesis that WIPK encoded a wounding-activated kinase of approximately 46 kDa. However, through the use of a WIPK-specific antibody, it was demonstrated that wounding induced little, if any, increases in WIPK protein and enzymatic activity levels, in contrast to a significant rise in its transcript level (41). Rather, WIPK was found to be activated by most of the pathogen-associated stimuli that activate SIPK, including the CWD elicitor, both elicitins, TMV infection, and the presence of Avr9 peptide in transgenic tobacco expressing Cf-9 (Table 1). These studies also revealed that WIPK activation is regulated at multiple levels. Some stimuli, such as the CWD elicitor, rapidly and transiently induced WIPK enzymatic activity to only low levels whereas the slower and more dramatic increase in its mRNA and protein levels did not result in higher enzymatic activity (43). On the other hand, the dramatic rise in WIPK activity after TMV infection or elicitin treatment was preceded by and required both increases in WIPK transcription and translation, as well as posttranslational phosphorylation (40, 43). By contrast, activation of SIPK, like that for MAP kinases in yeast and animals, was regulated strictly at the posttranslational level by dual phosphorylation of threonine and tyrosine residues, presumably in the conserved TEY motif located between subdomains VII and VIII of the kinase catalytic domain.